Yang et al. Molecular and Cellular Therapies 2014, 2:8 http://www.molcelltherapies.com/content/2/1/8
REVIEW
Open Access
Application of circulating tumor cells scope technique on circulating tumor cell research Dawei Yang1*, Lijie Wang2 and Xiaochen Tian3
Abstract Circulating tumor cells (CTCs) are becoming promising biomarkers in several cancers, such as colon, prostate, and breast carcinomas. Independent research groups have reported a correlation between CTC numbers and patient prognosis. Even more, the development of personalized medicine gives physicians impetus to utilize the advancement of molecular characterization of CTCs. This review introduces a new technique, CTCscope, and compares it with the current methods of CTCs detection, with particular emphasis on cancer research, and discusses the future application of this new method from bench to bed-side. Keywords: CTCs, ISH, qRT-PCR, CTCscope
Review Introduction
Circulating tumor cells (CTCs) are becoming promising biomarkers in several cancers, such as colon, prostate, and breast carcinomas. Due to the lost expression of common epithelial markers by certain types of CTCs, such as EpCAM or the cytokeratin (CK), current positive selection strategies are usually with low sensitivity and efficiency [1,2]. For example, most breast tumour stem cells are estrogen receptor negative, which could not be detected by positive selection method [3] (Table 1). In 2011, RNAscope or CTCscope was developed for detecting multiple tumour-specific marker mRNAs in tumor tissues [9,10], as well as CTCs from blood [11]. In brief, peripheral blood mononuclear cells (PBMC) including CTCs are enriched and then placed on the slides. After fixed by 10% formaldehyde solution, cells are hybridized with specifically designed target probes. The fluorescent signal is amplified by a series of nucleic acid hybridization and imaged by fluorescent microscopy for CTCscope analysis. The schematic of CTCscope assay procedure was showed in Figure 1. Compared to the current CTCs detection methods, CTCscope approach is more sensitive and allows single molecule detection in situ in individual cells by using a novel system * Correspondence: [email protected] 1 Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, No. 180 Fenglin Rd., Shanghai 200032, China Full list of author information is available at the end of the article
of probe design and signal amplification. This ability makes CTCscope a powerful platform in routine clinical assays for CTCs detection.
Tumorgenesis research
In vivo study, Burd et al. used standard homologous recombination procedures to target firefly luciferase on the SV40 polyadenylation site to exon 1α of the endogenous p16INK4a gene [12]. Then they applied RNA in situ hybridization (ISH) by using RNAscope 2.0 technology to detect this faithfully reports expression gene, which serves as a tumor suppressor and aging biomarker. Their work suggests that p16INK4a activation is a characteristic of all emerging cancers and it could be set as a sensitive, unbiased reporter of neoplastic transformation. While in situ RNA study, Staudt et al. applied macrophage inflammatory protein-1a/CCL3-specific RNA target probe set, which targeted nucleotides 23 to 771 of the CCL cDNA sequence to detect CCL3 mRNA in tumor samples [13]. CCL3 is a chemokine, which is known to amplify inflammation. They found inhibiting NF-ҡB reversed the increase in CCL3 expression associated with LDL receptor-related protein (LRP1) gene silencing in macrophage-like cells. LRP1 is a type 1 transmembrane receptor, which mediates ligand endocytosis and impact cell migration. This phenomenon suggested to prevent LRP1 down regulation in myeloid cells might suppress monocyte recruitment to tumors and cancer angiogenesis.
© 2014 Yang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Method
Enrichment
Detection
Sensitivity
Specificity Sample volume
Cell Cell morphology viability
Limitations
Advantages
Reference
CTCscope
Density of RNA ISH (Multiplex mononuclear cells CTC specific mRNA) (Ficoll centrifugation)
High
High
Good
Not easy to perform in a clinical lab
Simple technique; EpCAM-negative cells can be isolated
Payne et al. 2012 [4]
CellSearch
Immunomagnetic enrichment of EpCAM-positive cells
Easy to use semiautomated system; reproducibility; only assay approved by FDA
Peeters et al. 2013 [5]
AdnaTest
Detected only viable RNA isolation and High CK19-releasing multiplex PCR for tumour-specific transcripts (MUC1+/ HER2+/EpCAM+)
IHC (CKs, CD45, DAPI) Moderate; High low in EpCAM negative cases
Less blood sample
Live cells
At least Poor 7.5 mL blood sample
Live or Cannot identify dead cells EpCAM-negative CTCs (such as tumour stem cells with estrogen receptor negative phenotype in breast cancers); expensive;
High
5mL
-
MUC1 is also expressed on Enables the activated T lymphocytes; additional analysis Semiquantitative PCR of transcripts
Tewes et al. 2009 [6]
Talasaz et al. 2009 [7]
Good
MagSweeper Magnetic isolation
None
High
High
At least Good 7.5 mL blood sample
Live cells
Low efficiency
No impact on the transcriptional profile of single cancer cell isolated
Cytometric analysis
None
Low
-
-
Live cells
Dependent on expression of epithelial or tumor markers
Further characterization Lu et al. (FACS); multiple 2010 [8] antibodies; morphology evaluation
Immunoflurorescent detection of antigen expression
Good
Yang et al. Molecular and Cellular Therapies 2014, 2:8 http://www.molcelltherapies.com/content/2/1/8
Table 1 Comparison of current CTC enrichment and detection method with CTCscope technique
Page 2 of 5
Yang et al. Molecular and Cellular Therapies 2014, 2:8 http://www.molcelltherapies.com/content/2/1/8
Page 3 of 5
Blood drawing Primary Lesion
Metastasis Lesion
A Physical Procedure
CellSearch/ AdnaTest
ELISPOT
MagSweeper
Ephesia
Cytometry
CTCscope Peripheral Blood
PBMC
B
Ficoll
C
RBC RNAscope Procedure
D
Biological Procedure
Step 1
Tumor specific mRNA
RBC
Target Probe
Leukocytes
EpCam+
Laser
zz Step 2
Step 4
Magnet
Preamplifier CTC Amplifer Stromal cell
Step 3
Micropost
Label Probe
Figure 1 Enrichment of CTCs from peripheral blood of cancer patients by physical or biological properties. A: Transition of CTC from primary lesion to metastasis lesion. B: Physical properties include density (Ficoll centrifugation) - CTCscope. C: Biological properties are based on the following: the expression of cell surface markers, including an epithelial cell adhesion molecule (EpCAM) for positive selection – CellSearch/AdnaTest; anti-EpCAM antibodies conjugated with magnetic beads, for enriching CTCs in a magnetic field – MagSweeper; anti-EpCAM antibodies on microposts or columns of nanobeads – ELISPOT/Ephesia; anti-EpCAM antibodies conjugated to 3-μm beads to increase the size of CTCs before filtration - Cytometry. D: Schematic of the RNAscope assay procedure - CTCscope. In step 1, cells are fixed and permeabilized to allow for target probe access. In step 2, target RNA-specific oligonucleotide probes (Z) are hybridized in pairs (ZZ) to multiple RNA targets. In step 3, multiple signal amplification molecules are hybridized, each recognizing a specific target probe, and each unique label probe is conjugated to a different fluorophore or enzyme. In step 4, signals are detected using an epifluorescent microscope (for fluorescent label) (CTCscope image are reproduced from Payne et al. [4]) PBMC: peripheral blood mononuclear cells; CTC: circulating tumor cells; RBC: red blood cell.
EMT biomarkers development
Yu et al. optimized microfluidic capture of CTCs with epithelial- and tumor-specific antibodies, and they then applied this technology to analyze epithelial-mesenchymal transition (EMT) in CTCs from breast cancer patients [11]. They established quantitative, dual-colorimetric RNA-ISH assay to examine the expression of seven pooled epithelial (E) transcripts and three mesenchymal (M) transcripts in tumor cells. In their research, they found that all three major histological subtypes of invasive breast cancer (i.e. ER+/PR+ subtype; HER2+ subtype; and the ER–/PR–/ HER2– triple negative subtype) contained rare tumor cells with epithelial morphology that stained with both E and M markers, while that benign breast tissue and tumor cells in pre-invasive ductal carcinoma in situ (DCIS) lesions and reactive stromal cells were exclusively epithelial
or mesenchymal. By comparing CTC features in pre- and post treatment blood samples from clinical cases, they found patients who responded to therapy showed a decrease in CTC numbers and/or a proportional decrease in M + compared with E+ CTCs after treatment. In contrast, the patients who had progressive disease while on therapy showed an increased number of M+ CTCs in the post treatment samples. Cancer diagnostic research
Payne et al. applied a probe set against mRNAs encoding cytokeratin 8, 14, 17, 18, 19 and 20, EpCAM, and MUC1 (traditional epithelial cell markers) to detect epithelial cells and another probe set with different fluorescent color was used to detect three genes expressed in tumor cells that have undergone EMT (Twist, N-Cadherin, and
Yang et al. Molecular and Cellular Therapies 2014, 2:8 http://www.molcelltherapies.com/content/2/1/8
fibronectin) expressed in CTCs which have undergone EMT [4]. They found that breast cancer cells could be identified by strong pan-CK staining, whereas the surrounding PBMCs showed minimal fluorescent signals (Figure 1D). Also they found different EGFR mRNA expression levels consistent with the known EGFR protein expression status. The advance of application of fluorescence in situ hybridization technique is to detect the gene translocation. Tana et al. applied RNAscope formalin fixed paraffin embed (FFPE) assay in epithelioid hemangioendothelioma (EHE) tissue sections to detect the translocation (the chimeric WWTR1/CAMTA1 transcription factor), which would assist in the evaluation of this diagnostically challenging neoplasm as well as may represent a therapeutic target for EHE [14]. By multispectral imaging system, WWTR1/CAMTA1 fusion transcripts presented yellow punctate dots, while wild-type WWTR1 and CAMTA1 transcripts were stained with red or green punctate dots, respectively. Among these studies, it confirms that CTCscope provides additional prognostic and predictive information in therapy monitoring. Cancer stem cell research
Currently, In situ study, by RNAscope technology, groups of cancer stem cell biomarker was confirmed. Barry et al. applied RNAscope for Olfm4, which marks crypt base columnar (CBCs) stem cells in intestinal tissue, and to determine if intestinal stem cell (ISC) numbers was reduced by YAP (a protein known as its powerful growth-inducing and oncogenic properties) expression [15]. And in their research, they found epithelial-specific expression of YAP suppressed intestinal cell renewal, which occurred through inhibition of the Wnt-singaling pathway. Ziskin et al. also applied multiplex fluorescent ISH and chromogenic nonisotopic ISH in 57 colorectal adenocarcinomas to detect 19 putative intestinal stem cell markers [16]. They found the G protein-coupled receptor (lgr5) and intestinal stem cells signature gene (Ascl2) was expressed on mainly colorectal cancers, which supported the hypothesis that the cancer cells were derived from lgr5+/Ascl2+ crypt stem cells. Yan et al. applied fluorescence in situ hybridization (FISH) to detect lgr5 mRNA and genetic signature of panyellow fluorescent protein (YFP) expression within the Polycomb group protein (Bmi1) + clonally derived spheroids [17]. The result indicated that the Bmi1+ ISC lineage could generate Lgr5+ cells in vitro.
Page 4 of 5
nucleotides) and polyclonal rabbit anti-CD31 antibodies in a HA-SNV infection model [18]. They found that virus antigen was predominantly expressed in CD31-positive cells in lung tissues obtained from virus infected hamsters. Another virus research group, Ouwendijk et al. analyzed consecutive ganglionic sections in a reactivated Simian Varicella Virus (SVV) model, by ISH for SVV open reading frame 61 (ORF61) antisense RNA and IFNγ-inducible protein-10 (IP-10/CXCL10) transcripts [19]. The abundance of T cell clusters were correlated with CXCL10 RNA, but not with those of SVV ORF61 antisense RNA. So they concluded after SVV reactivation and transient T-cell infiltration, possibly medicated by CXCL10. Recently, Bishop et al. reported the development of RNA ISH probes complementary to E6/E7 mRNA, which permits direct visualization of human papillomavirus (HPV) transcripts, in routinely processed tissues and provide an accurate HPV detection method for the clinical physicians. Besides, they found p16 expression was strongly associated with the presence of HPV E6/E7 mRNA [20]. By comparison, they confirmed a high rate of concordance (99%) between the E6/E7 mRNA method and HPV DNA ISH.
Conclusions This recently developed new technique, CTCscope, is designed for detection mRNA expression on CTCs. Due to its advantage of highly sensitivity, RNA ISH empower its high sensitivity and specificity to define low expression of commonly used biomarkers. However the future application of CTCscope from bench to bed-side seems both promising and challenged. Certain limitations of this approach may hamper its clinical use, e.g. the assay protocol which contains multiple steps is not easy to perform in a clinical lab, the changing of hybridization probes against the target mRNA may reduce the reliability of the detection, and the cut off value and clinical significance of CTC numbers need to be determined for different cancers. Abbreviations CTCs: Circulating tumor cells; CBCs: Crypt base columnar; DCIS: Ductal carcinoma in situ; E: Epithelial; EMT: Epithelial-mesenchymal transition; EHE: Epithelioid hemangioendothelioma; FISH: Fluorescence in situ hybridization; FFEP: Formalin fixed paraffin embed; HA-SNV: Hamster-adapted Sin Nombre Virus; HPV: Human papillomavirus; ISH: In situ hybridization; ISC: Intestinal stem cell; M: Mesenchymal; ORF61: Open reading frame 61; SVV: Simian Varicella Virus.
Other application
On several virus infection models, CTCscope became a popular research tool for detection certain mRNA expression. In order to confirm Hamster-adapted Sin Nombre Virus (HA-SNV) replication in pulmonary endothelial cells, Safronetz et al. applied monoclonal antibodies targeting the nucleoprotein (anti-SNV NP clone 5 F1/F7)
Competing interests The authors, Dawei Yang, Lijie Wang and Xiaochen Tian declare that they have no competing interests. The work is funded by Shanghai Leading Academic Discipline Project, National Key Scientific & Technology Support Program and Shanghai Science & Technology Committee. The sponsor had no influence on the study design, information collection, interpretation and the writing of the manuscript.
Yang et al. Molecular and Cellular Therapies 2014, 2:8 http://www.molcelltherapies.com/content/2/1/8
Authors’ contribution All authors participated in the work and made substantial contributions to conception, literature research and paper writing. They devoted a lot to the data collection and interpretation. DY led the drafting of the article. LW modified the manuscript. XT carried out literature research. All authors participated in critical revisions, have granted final approval of the final manuscript and agree to be accountable for all aspects of the work. Acknowledgements This work was supported by Shanghai Leading Academic Discipline Project (No. B115), National Key Scientific & Technology Support Program (No. 2013BAI09B09) and Shanghai Science & Technology Committee (No. 13ZR1452400). Author details 1 Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, No. 180 Fenglin Rd., Shanghai 200032, China. 2Department of Health Service Management, Second Military Medical University, No. 800 Xiangyin Rd., Shanghai 200433, China. 3China Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, No. 138 Yixueyuan Rd., Shanghai 200032, China. Received: 16 October 2013 Accepted: 10 February 2014 Published: 10 March 2014 References 1. Liu Z, Fusi A, Klopocki E, Schmittel A, Tinhofer I, Nonnenmacher A, Keilholz U: Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients. J Transl Med 2011, 9:70. 2. Cen P, Ni X, Yang J, Graham DY, Li M: Circulating tumor cells in the diagnosis and management of pancreatic cancer. Biochim Biophys Acta 1826, 2012:350–356. 3. Eden JA: Human breast cancer stem cells and sex hormones–a narrative review. Menopause 2010, 17:801–810. 4. Payne RE, Wang F, Su N, Krell J, Zebrowski A, Yagüe E, Ma XJ, Luo Y, Coombes RC: Viable circulating tumour cell detection using multiplex RNA in situ hybridisation predicts progression-free survival in metastatic breast cancer patients. Br J Cancer 2012, 106:1790–1797. 5. Peeters DJ, van Dam PJ, Van den Eynden GG, Rutten A, Wuyts H, Pouillon L, Peeters M, Pauwels P, Van Laere SJ, van Dam PA, Vermeulen PB, Dirix LY: Detection and prognostic significance of circulating tumour cells in patients with metastatic breast cancer according to immunohistochemical subtypes. Br J Cancer 2013. doi:10.1038/bjc.2013.743. [Epub ahead of print]. 6. Tewes M, Aktas B, Welt A, Mueller S, Hauch S, Kimmig R, Kasimir-Bauer S: Molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer: an option for monitoring response to breast cancer related therapies. Breast Cancer Res Treat 2009, 115:581–590. 7. Talasaz AH, Powell AA, Huber DE, Berbee JG, Roh KH, Yu W, Xiao W, Davis MM, Pease RF, Mindrinos MN, Jeffrey SS, Davis RW: Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device. Proc Natl Acad Sci USA 2009, 106:3970–3975. 8. Lu J, Fan T, Zhao Q, Zeng W, Zaslavsky E, Chen JJ, Frohman MA, Golightly MG, Madajewicz S, Chen WT: Isolation of circulating epithelial and tumor progenitor cells with an invasive phenotype from breast cancer patients. Int J Cancer 2010, 126:669–683. 9. Wang F, Flanagan J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, Luo Y: RNAscope: a novel in situ RNA analysis platform for formalinfixed, paraffin-embedded tissues. J Mol Diagn 2012, 14:22–29. 10. Ukpo OC, Flanagan JJ, Ma XJ, Luo Y, Thorstad WL, Lewis JS Jr: High-risk human papillomavirus E6/E7 mRNA detection by a novel in situ hybridization assay strongly correlates with p16 expression and patient outcomes in oropharyngeal squamous cell carcinoma. Am J Surg Pathol 2011, 35:1343–1350. 11. Yu M, Bardia A, Wittner BS, Stott SL, Smas ME, Ting DT, Isakoff SJ, Ciciliano JC, Wells MN, Shah AM, Concannon KF, Donaldson MC, Sequist LV, Brachtel E, Sgroi D, Baselga J, Ramaswamy S, Toner M, Haber DA, Maheswaran S: Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science 2013, 339:580–584.
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12. Burd CE, Sorrentino JA, Clark KS, Darr DB, Krishnamurthy J, Deal AM, Bardeesy N, Castrillon DH, Beach DH, Sharpless NE: Monitoring tumorigenesis and senescence in vivo with a p16(INK4a)-luciferase model. Cell 2013, 152:340–351. 13. Staudt ND, Jo M, Hu J, Bristow JM, Pizzo DP, Gaultier A, VandenBerg SR, Gonias SL: Myeloid cell receptor LRP1/CD91 regulates monocyte recruitment and angiogenesis in tumors. Cancer Res 2013, 73:3902–3912. 14. Tanas MR, Sboner A, Oliveira AM, Erickson-Johnson MR, Hespelt J, Hanwright PJ, Flanagan J, Luo Y, Fenwick K, Natrajan R, Mitsopoulos C, Zvelebil M, Hoch BL, Weiss SW, Debiec-Rychter M, Sciot R, West RB, Lazar AJ, Ashworth A, Reis-Filho JS, Lord CJ, Gerstein MB, Rubin MA, Rubin BP: Identification of a disease-defining gene fusion in epithelioid hemangioendothelioma. Sci Transl Med 2011, 3:98ra82. 15. Barry ER, Morikawa T, Butler BL, Shrestha K, de la Rosa R, Yan KS, Fuchs CS, Magness ST, Smits R, Ogino S, Kuo CJ, Camargo FD: Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature 2013, 493:106–110. 16. Ziskin JL, Dunlap D, Yaylaoglu M, Fodor IK, Forrest WF, Patel R, Ge N, Hutchins GG, Pine JK, Quirke P, Koeppen H, Jubb AM: In situ validation of an intestinal stem cell signature in colorectal cancer. Gut 2012, 62:1012–1023. 17. Yan KS, Chia LA, Li X, Ootani A, Su J, Lee JY, Su N, Luo Y, Heilshorn SC, Amieva MR, Sangiorgi E, Capecchi MR, Kuo CJ: The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc Nati Acad Sci 2012, 109:466–471. 18. Safronetz D, Prescott J, Haddock E, Scott DP, Feldmann H, Ebihara H: Hamster-adapted Sin Nombre virus causes disseminated infection and efficiently replicates in pulmonary endothelial cells without signs of disease. J Virol 2013, 87:4778–4782. 19. Ouwendijk WJ, Abendroth A, Traina-Dorge V, Getu S, Steain M, Wellish M, Andeweg AC, Osterhaus AD, Gilden D, Verjans GM, Mahalingam R: T-cell infiltration correlates with CXCL10 expression in ganglia of cynomolgus macaques with reactivated simian varicella virus. J Virol 2013, 87:2979–2982. 20. Bishop JA, Ma XJ, Wang H, Luo Y, Illei PB, Begum S, Taube JM, Koch WM, Westra WH: Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method. Am J Surg Pathol 2012, 36:1874–1882. doi:10.1186/2052-8426-2-8 Cite this article as: Yang et al.: Application of circulating tumor cells scope technique on circulating tumor cell research. Molecular and Cellular Therapies 2014 2:8.
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REVIEW
Open Access
Application of circulating tumor cells scope technique on circulating tumor cell research Dawei Yang1*, Lijie Wang2 and Xiaochen Tian3
Abstract Circulating tumor cells (CTCs) are becoming promising biomarkers in several cancers, such as colon, prostate, and breast carcinomas. Independent research groups have reported a correlation between CTC numbers and patient prognosis. Even more, the development of personalized medicine gives physicians impetus to utilize the advancement of molecular characterization of CTCs. This review introduces a new technique, CTCscope, and compares it with the current methods of CTCs detection, with particular emphasis on cancer research, and discusses the future application of this new method from bench to bed-side. Keywords: CTCs, ISH, qRT-PCR, CTCscope
Review Introduction
Circulating tumor cells (CTCs) are becoming promising biomarkers in several cancers, such as colon, prostate, and breast carcinomas. Due to the lost expression of common epithelial markers by certain types of CTCs, such as EpCAM or the cytokeratin (CK), current positive selection strategies are usually with low sensitivity and efficiency [1,2]. For example, most breast tumour stem cells are estrogen receptor negative, which could not be detected by positive selection method [3] (Table 1). In 2011, RNAscope or CTCscope was developed for detecting multiple tumour-specific marker mRNAs in tumor tissues [9,10], as well as CTCs from blood [11]. In brief, peripheral blood mononuclear cells (PBMC) including CTCs are enriched and then placed on the slides. After fixed by 10% formaldehyde solution, cells are hybridized with specifically designed target probes. The fluorescent signal is amplified by a series of nucleic acid hybridization and imaged by fluorescent microscopy for CTCscope analysis. The schematic of CTCscope assay procedure was showed in Figure 1. Compared to the current CTCs detection methods, CTCscope approach is more sensitive and allows single molecule detection in situ in individual cells by using a novel system * Correspondence: [email protected] 1 Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, No. 180 Fenglin Rd., Shanghai 200032, China Full list of author information is available at the end of the article
of probe design and signal amplification. This ability makes CTCscope a powerful platform in routine clinical assays for CTCs detection.
Tumorgenesis research
In vivo study, Burd et al. used standard homologous recombination procedures to target firefly luciferase on the SV40 polyadenylation site to exon 1α of the endogenous p16INK4a gene [12]. Then they applied RNA in situ hybridization (ISH) by using RNAscope 2.0 technology to detect this faithfully reports expression gene, which serves as a tumor suppressor and aging biomarker. Their work suggests that p16INK4a activation is a characteristic of all emerging cancers and it could be set as a sensitive, unbiased reporter of neoplastic transformation. While in situ RNA study, Staudt et al. applied macrophage inflammatory protein-1a/CCL3-specific RNA target probe set, which targeted nucleotides 23 to 771 of the CCL cDNA sequence to detect CCL3 mRNA in tumor samples [13]. CCL3 is a chemokine, which is known to amplify inflammation. They found inhibiting NF-ҡB reversed the increase in CCL3 expression associated with LDL receptor-related protein (LRP1) gene silencing in macrophage-like cells. LRP1 is a type 1 transmembrane receptor, which mediates ligand endocytosis and impact cell migration. This phenomenon suggested to prevent LRP1 down regulation in myeloid cells might suppress monocyte recruitment to tumors and cancer angiogenesis.
© 2014 Yang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Method
Enrichment
Detection
Sensitivity
Specificity Sample volume
Cell Cell morphology viability
Limitations
Advantages
Reference
CTCscope
Density of RNA ISH (Multiplex mononuclear cells CTC specific mRNA) (Ficoll centrifugation)
High
High
Good
Not easy to perform in a clinical lab
Simple technique; EpCAM-negative cells can be isolated
Payne et al. 2012 [4]
CellSearch
Immunomagnetic enrichment of EpCAM-positive cells
Easy to use semiautomated system; reproducibility; only assay approved by FDA
Peeters et al. 2013 [5]
AdnaTest
Detected only viable RNA isolation and High CK19-releasing multiplex PCR for tumour-specific transcripts (MUC1+/ HER2+/EpCAM+)
IHC (CKs, CD45, DAPI) Moderate; High low in EpCAM negative cases
Less blood sample
Live cells
At least Poor 7.5 mL blood sample
Live or Cannot identify dead cells EpCAM-negative CTCs (such as tumour stem cells with estrogen receptor negative phenotype in breast cancers); expensive;
High
5mL
-
MUC1 is also expressed on Enables the activated T lymphocytes; additional analysis Semiquantitative PCR of transcripts
Tewes et al. 2009 [6]
Talasaz et al. 2009 [7]
Good
MagSweeper Magnetic isolation
None
High
High
At least Good 7.5 mL blood sample
Live cells
Low efficiency
No impact on the transcriptional profile of single cancer cell isolated
Cytometric analysis
None
Low
-
-
Live cells
Dependent on expression of epithelial or tumor markers
Further characterization Lu et al. (FACS); multiple 2010 [8] antibodies; morphology evaluation
Immunoflurorescent detection of antigen expression
Good
Yang et al. Molecular and Cellular Therapies 2014, 2:8 http://www.molcelltherapies.com/content/2/1/8
Table 1 Comparison of current CTC enrichment and detection method with CTCscope technique
Page 2 of 5
Yang et al. Molecular and Cellular Therapies 2014, 2:8 http://www.molcelltherapies.com/content/2/1/8
Page 3 of 5
Blood drawing Primary Lesion
Metastasis Lesion
A Physical Procedure
CellSearch/ AdnaTest
ELISPOT
MagSweeper
Ephesia
Cytometry
CTCscope Peripheral Blood
PBMC
B
Ficoll
C
RBC RNAscope Procedure
D
Biological Procedure
Step 1
Tumor specific mRNA
RBC
Target Probe
Leukocytes
EpCam+
Laser
zz Step 2
Step 4
Magnet
Preamplifier CTC Amplifer Stromal cell
Step 3
Micropost
Label Probe
Figure 1 Enrichment of CTCs from peripheral blood of cancer patients by physical or biological properties. A: Transition of CTC from primary lesion to metastasis lesion. B: Physical properties include density (Ficoll centrifugation) - CTCscope. C: Biological properties are based on the following: the expression of cell surface markers, including an epithelial cell adhesion molecule (EpCAM) for positive selection – CellSearch/AdnaTest; anti-EpCAM antibodies conjugated with magnetic beads, for enriching CTCs in a magnetic field – MagSweeper; anti-EpCAM antibodies on microposts or columns of nanobeads – ELISPOT/Ephesia; anti-EpCAM antibodies conjugated to 3-μm beads to increase the size of CTCs before filtration - Cytometry. D: Schematic of the RNAscope assay procedure - CTCscope. In step 1, cells are fixed and permeabilized to allow for target probe access. In step 2, target RNA-specific oligonucleotide probes (Z) are hybridized in pairs (ZZ) to multiple RNA targets. In step 3, multiple signal amplification molecules are hybridized, each recognizing a specific target probe, and each unique label probe is conjugated to a different fluorophore or enzyme. In step 4, signals are detected using an epifluorescent microscope (for fluorescent label) (CTCscope image are reproduced from Payne et al. [4]) PBMC: peripheral blood mononuclear cells; CTC: circulating tumor cells; RBC: red blood cell.
EMT biomarkers development
Yu et al. optimized microfluidic capture of CTCs with epithelial- and tumor-specific antibodies, and they then applied this technology to analyze epithelial-mesenchymal transition (EMT) in CTCs from breast cancer patients [11]. They established quantitative, dual-colorimetric RNA-ISH assay to examine the expression of seven pooled epithelial (E) transcripts and three mesenchymal (M) transcripts in tumor cells. In their research, they found that all three major histological subtypes of invasive breast cancer (i.e. ER+/PR+ subtype; HER2+ subtype; and the ER–/PR–/ HER2– triple negative subtype) contained rare tumor cells with epithelial morphology that stained with both E and M markers, while that benign breast tissue and tumor cells in pre-invasive ductal carcinoma in situ (DCIS) lesions and reactive stromal cells were exclusively epithelial
or mesenchymal. By comparing CTC features in pre- and post treatment blood samples from clinical cases, they found patients who responded to therapy showed a decrease in CTC numbers and/or a proportional decrease in M + compared with E+ CTCs after treatment. In contrast, the patients who had progressive disease while on therapy showed an increased number of M+ CTCs in the post treatment samples. Cancer diagnostic research
Payne et al. applied a probe set against mRNAs encoding cytokeratin 8, 14, 17, 18, 19 and 20, EpCAM, and MUC1 (traditional epithelial cell markers) to detect epithelial cells and another probe set with different fluorescent color was used to detect three genes expressed in tumor cells that have undergone EMT (Twist, N-Cadherin, and
Yang et al. Molecular and Cellular Therapies 2014, 2:8 http://www.molcelltherapies.com/content/2/1/8
fibronectin) expressed in CTCs which have undergone EMT [4]. They found that breast cancer cells could be identified by strong pan-CK staining, whereas the surrounding PBMCs showed minimal fluorescent signals (Figure 1D). Also they found different EGFR mRNA expression levels consistent with the known EGFR protein expression status. The advance of application of fluorescence in situ hybridization technique is to detect the gene translocation. Tana et al. applied RNAscope formalin fixed paraffin embed (FFPE) assay in epithelioid hemangioendothelioma (EHE) tissue sections to detect the translocation (the chimeric WWTR1/CAMTA1 transcription factor), which would assist in the evaluation of this diagnostically challenging neoplasm as well as may represent a therapeutic target for EHE [14]. By multispectral imaging system, WWTR1/CAMTA1 fusion transcripts presented yellow punctate dots, while wild-type WWTR1 and CAMTA1 transcripts were stained with red or green punctate dots, respectively. Among these studies, it confirms that CTCscope provides additional prognostic and predictive information in therapy monitoring. Cancer stem cell research
Currently, In situ study, by RNAscope technology, groups of cancer stem cell biomarker was confirmed. Barry et al. applied RNAscope for Olfm4, which marks crypt base columnar (CBCs) stem cells in intestinal tissue, and to determine if intestinal stem cell (ISC) numbers was reduced by YAP (a protein known as its powerful growth-inducing and oncogenic properties) expression [15]. And in their research, they found epithelial-specific expression of YAP suppressed intestinal cell renewal, which occurred through inhibition of the Wnt-singaling pathway. Ziskin et al. also applied multiplex fluorescent ISH and chromogenic nonisotopic ISH in 57 colorectal adenocarcinomas to detect 19 putative intestinal stem cell markers [16]. They found the G protein-coupled receptor (lgr5) and intestinal stem cells signature gene (Ascl2) was expressed on mainly colorectal cancers, which supported the hypothesis that the cancer cells were derived from lgr5+/Ascl2+ crypt stem cells. Yan et al. applied fluorescence in situ hybridization (FISH) to detect lgr5 mRNA and genetic signature of panyellow fluorescent protein (YFP) expression within the Polycomb group protein (Bmi1) + clonally derived spheroids [17]. The result indicated that the Bmi1+ ISC lineage could generate Lgr5+ cells in vitro.
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nucleotides) and polyclonal rabbit anti-CD31 antibodies in a HA-SNV infection model [18]. They found that virus antigen was predominantly expressed in CD31-positive cells in lung tissues obtained from virus infected hamsters. Another virus research group, Ouwendijk et al. analyzed consecutive ganglionic sections in a reactivated Simian Varicella Virus (SVV) model, by ISH for SVV open reading frame 61 (ORF61) antisense RNA and IFNγ-inducible protein-10 (IP-10/CXCL10) transcripts [19]. The abundance of T cell clusters were correlated with CXCL10 RNA, but not with those of SVV ORF61 antisense RNA. So they concluded after SVV reactivation and transient T-cell infiltration, possibly medicated by CXCL10. Recently, Bishop et al. reported the development of RNA ISH probes complementary to E6/E7 mRNA, which permits direct visualization of human papillomavirus (HPV) transcripts, in routinely processed tissues and provide an accurate HPV detection method for the clinical physicians. Besides, they found p16 expression was strongly associated with the presence of HPV E6/E7 mRNA [20]. By comparison, they confirmed a high rate of concordance (99%) between the E6/E7 mRNA method and HPV DNA ISH.
Conclusions This recently developed new technique, CTCscope, is designed for detection mRNA expression on CTCs. Due to its advantage of highly sensitivity, RNA ISH empower its high sensitivity and specificity to define low expression of commonly used biomarkers. However the future application of CTCscope from bench to bed-side seems both promising and challenged. Certain limitations of this approach may hamper its clinical use, e.g. the assay protocol which contains multiple steps is not easy to perform in a clinical lab, the changing of hybridization probes against the target mRNA may reduce the reliability of the detection, and the cut off value and clinical significance of CTC numbers need to be determined for different cancers. Abbreviations CTCs: Circulating tumor cells; CBCs: Crypt base columnar; DCIS: Ductal carcinoma in situ; E: Epithelial; EMT: Epithelial-mesenchymal transition; EHE: Epithelioid hemangioendothelioma; FISH: Fluorescence in situ hybridization; FFEP: Formalin fixed paraffin embed; HA-SNV: Hamster-adapted Sin Nombre Virus; HPV: Human papillomavirus; ISH: In situ hybridization; ISC: Intestinal stem cell; M: Mesenchymal; ORF61: Open reading frame 61; SVV: Simian Varicella Virus.
Other application
On several virus infection models, CTCscope became a popular research tool for detection certain mRNA expression. In order to confirm Hamster-adapted Sin Nombre Virus (HA-SNV) replication in pulmonary endothelial cells, Safronetz et al. applied monoclonal antibodies targeting the nucleoprotein (anti-SNV NP clone 5 F1/F7)
Competing interests The authors, Dawei Yang, Lijie Wang and Xiaochen Tian declare that they have no competing interests. The work is funded by Shanghai Leading Academic Discipline Project, National Key Scientific & Technology Support Program and Shanghai Science & Technology Committee. The sponsor had no influence on the study design, information collection, interpretation and the writing of the manuscript.
Yang et al. Molecular and Cellular Therapies 2014, 2:8 http://www.molcelltherapies.com/content/2/1/8
Authors’ contribution All authors participated in the work and made substantial contributions to conception, literature research and paper writing. They devoted a lot to the data collection and interpretation. DY led the drafting of the article. LW modified the manuscript. XT carried out literature research. All authors participated in critical revisions, have granted final approval of the final manuscript and agree to be accountable for all aspects of the work. Acknowledgements This work was supported by Shanghai Leading Academic Discipline Project (No. B115), National Key Scientific & Technology Support Program (No. 2013BAI09B09) and Shanghai Science & Technology Committee (No. 13ZR1452400). Author details 1 Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, No. 180 Fenglin Rd., Shanghai 200032, China. 2Department of Health Service Management, Second Military Medical University, No. 800 Xiangyin Rd., Shanghai 200433, China. 3China Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, No. 138 Yixueyuan Rd., Shanghai 200032, China. Received: 16 October 2013 Accepted: 10 February 2014 Published: 10 March 2014 References 1. Liu Z, Fusi A, Klopocki E, Schmittel A, Tinhofer I, Nonnenmacher A, Keilholz U: Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients. J Transl Med 2011, 9:70. 2. Cen P, Ni X, Yang J, Graham DY, Li M: Circulating tumor cells in the diagnosis and management of pancreatic cancer. Biochim Biophys Acta 1826, 2012:350–356. 3. Eden JA: Human breast cancer stem cells and sex hormones–a narrative review. Menopause 2010, 17:801–810. 4. Payne RE, Wang F, Su N, Krell J, Zebrowski A, Yagüe E, Ma XJ, Luo Y, Coombes RC: Viable circulating tumour cell detection using multiplex RNA in situ hybridisation predicts progression-free survival in metastatic breast cancer patients. Br J Cancer 2012, 106:1790–1797. 5. Peeters DJ, van Dam PJ, Van den Eynden GG, Rutten A, Wuyts H, Pouillon L, Peeters M, Pauwels P, Van Laere SJ, van Dam PA, Vermeulen PB, Dirix LY: Detection and prognostic significance of circulating tumour cells in patients with metastatic breast cancer according to immunohistochemical subtypes. Br J Cancer 2013. doi:10.1038/bjc.2013.743. [Epub ahead of print]. 6. Tewes M, Aktas B, Welt A, Mueller S, Hauch S, Kimmig R, Kasimir-Bauer S: Molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer: an option for monitoring response to breast cancer related therapies. Breast Cancer Res Treat 2009, 115:581–590. 7. Talasaz AH, Powell AA, Huber DE, Berbee JG, Roh KH, Yu W, Xiao W, Davis MM, Pease RF, Mindrinos MN, Jeffrey SS, Davis RW: Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device. Proc Natl Acad Sci USA 2009, 106:3970–3975. 8. Lu J, Fan T, Zhao Q, Zeng W, Zaslavsky E, Chen JJ, Frohman MA, Golightly MG, Madajewicz S, Chen WT: Isolation of circulating epithelial and tumor progenitor cells with an invasive phenotype from breast cancer patients. Int J Cancer 2010, 126:669–683. 9. Wang F, Flanagan J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, Luo Y: RNAscope: a novel in situ RNA analysis platform for formalinfixed, paraffin-embedded tissues. J Mol Diagn 2012, 14:22–29. 10. Ukpo OC, Flanagan JJ, Ma XJ, Luo Y, Thorstad WL, Lewis JS Jr: High-risk human papillomavirus E6/E7 mRNA detection by a novel in situ hybridization assay strongly correlates with p16 expression and patient outcomes in oropharyngeal squamous cell carcinoma. Am J Surg Pathol 2011, 35:1343–1350. 11. Yu M, Bardia A, Wittner BS, Stott SL, Smas ME, Ting DT, Isakoff SJ, Ciciliano JC, Wells MN, Shah AM, Concannon KF, Donaldson MC, Sequist LV, Brachtel E, Sgroi D, Baselga J, Ramaswamy S, Toner M, Haber DA, Maheswaran S: Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science 2013, 339:580–584.
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